title: Identification of palmitoyl protein thioesterase 1 in human THP1 monocytes and macrophages and characterization of unique biochemical activities for this enzyme.
abstract: The profiles of serine hydrolases in human and mouse macrophages are similar yet different. For instance, human macrophages express high levels of carboxylesterase 1 (CES1), whereas mouse macrophages have minimal amounts of the orthologous murine CES1. On the other hand, macrophages from both species exhibit limited expression of the canonical 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, MAGL. Our previous study showed CES1 was partly responsible for the hydrolysis of 2-AG (50%) and prostaglandin glyceryl esters (PG-Gs) (80-95%) in human THP1 monocytes and macrophages. However, MAGL and other endocannabinoid hydrolases, FAAH, ABHD6, and ABHD12, did not have a role because of limited expression or no expression. Thus, another enzyme was hypothesized to be responsible for the remaining 2-AG hydrolysis activity following chemical inhibition and immunodepletion of CES1 (previous study) or CES1 gene knockdown (this study). Here we identified two candidate serine hydrolases in THP1 cell lysates by activity-based protein profiling (ABPP)-MUDPIT and Western blotting: cathepsin G and palmitoyl protein thioesterase 1 (PPT1). Both proteins exhibited electrophoretic properties similar to those of a serine hydrolase in THP1 cells detected by gel-based ABPP at 31-32 kDa; however, only PPT1 exhibited lipolytic activity and hydrolyzed 2-AG in vitro. Interestingly, PPT1 was strongly expressed in THP1 cells but was significantly less reactive than cathepsin G toward the activity-based probe, fluorophosphonate-biotin. KIAA1363, another serine hydrolase, was also identified in THP1 cells but did not have significant lipolytic activity. On the basis of chemoproteomic profiling, immunodepletion studies, and chemical inhibitor profiles, we estimated that PPT1 contributed 32-40% of 2-AG hydrolysis activity in the THP1 cell line. In addition, pure recombinant PPT1 catalyzed the hydrolysis of 2-AG, PGE2-G, and PGF2α-G, although the catalytic efficiency of hydrolysis of 2-AG by PPT1 was ~10-fold lower than that of CES1. PPT1 was also insensitive to several chemical inhibitors that potently inhibit CES1, such as organophosphate poisons and JZL184. This is the first report to document the expression of PPT1 in a human monocyte and macrophage cell line and to show PPT1 can hydrolyze the natural substrates 2-AG and PG-Gs. These findings suggest that PPT1 may participate in endocannabinoid metabolism within specific cellular contexts and highlights the functional redundancy often exhibited by enzymes involved in lipid metabolism.
authors.affiliation: Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University , University, Mississippi 39762, United States.
authors.forename: Ran
authors.initials: R
authors.lastname: Wang
authors.forename: Abdolsamad
authors.initials: A
authors.lastname: Borazjani
authors.forename: Anberitha T
authors.initials: AT
authors.lastname: Matthews
authors.forename: Lee C
authors.initials: LC
authors.lastname: Mangum
authors.forename: Mariola J
authors.initials: MJ
authors.lastname: Edelmann
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Ross
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D016466:CHO Cells; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D063388:Endocannabinoids / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005989:Glycerides / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D006868:Hydrolysis; D008264:Macrophages / Q000166:cytology / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009000:Monocytes / Q000166:cytology / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005989:Glycerides; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C094503:glyceryl 2-arachidonate; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human; D013869:Thiolester Hydrolases; C416054:PPT1 protein, human; C095299:palmitoyl-protein thioesterase